Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. Zymomonas mobilis is a bacterial ethanologen that grows on glucose, fructose, and sucrose, metabolizing these sugars to CO2 and ethanol via the Entner-Douderoff pathway.
It is desirable to use hydrolyzed lignocellulosic biomass which can provide an abundantly available, low cost carbon substrate for use in fermentation for ethanol production. Xylose is the major pentose in hydrolyzed lignocellulosic materials. Though wild type strains of Z. mobilis cannot use xylose as a carbon source, recombinant strains that are able to grow on this sugar have been engineered (U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, WO 95/28476, Feldmann et al. (1992) Appl Microbiol Biotechnol 38: 354-361, Zhang et al. (1995) Science 267:240-243). These strains are modified for expression of four enzymes needed for xylose metabolism: 1) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase (U.S. Pat. No. 5,514,583, U.S. Pat. No. 6,566,107; Zhang et al. (1995) Science 267:240-243). Equipped with these four enzymes and the cell's normal metabolic machinery, three molecules of xylose are converted to two molecules of glucose 6-phosphate and one molecule of glyceraldehyde 3-phosphate, which are subsequently converted to ethanol and CO2 on the glucose side of the pathway (FIG. 1).
Though there has been success in engineering Z. mobilis strains for xylose metabolism, the strains do not grow and produce ethanol as well on xylose as on glucose. Even under ideal circumstances, xylose metabolism is 3- to 4-fold slower than glucose metabolism (Lawford et al. (2000) Applied Biochemistry and Biotechnology 84-86: 277-293), and the difference becomes much greater under adverse conditions. Because of the slow carbon flux, the steady-state level of ATP is also lower with growth on xylose (Kim et al. (2000) Applied and Environmental Microbiology 66(1):186-193), and as a result Z. mobilis is far more susceptible to stress and inhibitors when it is grown on this sugar (Joachimsthal et al. (2000) Applied Biochemistry and Biotechnology 84-86:343-356; Kim et al. (2000) Applied Biochemistry and Biotechnology 84-6:357-370). A particular stress encountered in using hydrolyzed lignocellulosic biomass for fermentation is the presence of acetate (Kim et al. (2000) Applied Biochemistry and Biotechnology 84-86:357-370), which is released from the acetylated xylose residues in hemicellulose during pre-treatment and saccharification processes.
Mechanisms for Z. mobilis to cope with stress related to acetate and other organic acids remain to be elucidated, and there are no reports in the literature about the genes that play a role in this process. Using rational design to genetically engineer a strain that has higher resistance to acetate is therefore currently not an option. On the other hand, Z. mobilis mutants that have greater tolerance for acetate have been described (Joachimsthal et al. (1998) Biotechnol. Lett. 20(2):137-142; Jeon et al. (2002) Biotechnol. Lett. 24:819-824; US Patent Application 20030162271). Selection after random chemical mutagenesis with nitrosoguanidine (NTG) was used to generate these mutants, but the modified genes that were responsible for the acetate-resistant phenotype were not identified in any of these cases. It was also not determined whether one mutation or multiple mutations were required for better fermentation performance in the presence of acetate. Thus it is currently not known from the studies cited above how to impart acetate tolerance to other strains of Z. mobilis using targeted genetic engineering.
There remains a need to identify genes involved in acetate tolerance that can be modified to produce acetate tolerant strains of Zymomonas for fermentation of hydrolysate, produced from pretreated and saccharified lignocellulosic biomass, to produce ethanol.